DESCRIPTION: Introns are removed from pre-mRNAs via two phosphodiester cleavage reactions catalyzed by spliceosomes. In contrast to most enzyme complexes, the spliceosome consists of more than one hundred components and is assembled de novo around each substrate. The substrates are diverse in size and sequence, containing many potential splice sites, yet the correct splice sites are invariably chosen. Splicing appears to be fundamentally RNA-catalyzed because the same chemical reactions occur in vitro for self-splicing Group II introns, in an RNA-only reaction. Spliceosome is formed through an ordered interaction of five snRNAs and a pre-mRNA substrate. The dynamic process of spliceosome assembly is catalyzed by a family of RNA -dependent ATPases explaining, at least in part the requirement for ATP hydrolysis in the spliceosomal reaction. Within this framework the specific aims of this application are as follows: i) employing the S. Cerevisiae system, a search for new spliceosomal proteins will be carried out. The proteins will be identified through a large genetic screen and by characterization of snRNAs and spliceosomal intermediates, ii) at least five spliceosomal RNA-dependent ATPases mediate different steps in spliceosome assembly, and yet in no case is the function of the ATP-driven reaction understood. Therefore, it is planned to determine the three dimensional structure of the DbpA protein from E. coli, a prototype of this family of proteins, whose target is a 73-nucleotide fragment of 23S rRNA. In addition, the roles of two RNA-dependent ATPases from yeast Prp5, which participates in pre-spliceosome assembly, and Prp22, which acts to release spliced mRNA product from the spliceosome will be identified, iii) concerning the roles of the snRNAs in catalysis, the principle questions to be answered are what interactions are in effect at the time of the first reaction, and what structural changes take place to allow the second reaction. When in spliceosome assembly do these interactions occur? What RNA-protein interactions mediate the formation of and stabilization of the active site? Using an in vitro reconstitution system Dr. Abelson will explore the chemical and structural requirements of functional snRNAs by screening specific base changes and deletions, base analogs, and backbone perturbations. By photo cross-linking methods, RNA-RNA and RNA-protein interactions in the spliceosome will be identified. He will determine whether cross-links are formed at successive steps of spliceosome assembly through the use of mutant extracts that block a particular step.